It has been demonstrated that tumor reactive T-cells can induce tumor regression in patients with metastatic melanoma and some of the target antigens have been identified. Still, the clinical impact of current immunomodulatory approaches by use of cytokines/biologic response modifiers, vaccination or the use of adoptive immunotherapy remains limited. We have recently identified and cloned the T-cell receptor (TCR) of a CD4 Tumor infiltrating lymphocyte with MHC class I restricted recognition and strong affinity for its target, the 368-376 peptide of tyrosinase. The overall aim of this proposal is to develop an adoptive immunotherapy approach using TCR-gene modified PBL. In specific aim 1 we will determine if the retroviral vector encoding the TCR from an HLA-A2 restricted, tryosinase:368-376 reactive T cell clone (TIL 13831) can engineer normal peripheral blood T cells from normal donors and melanoma patients to recognize melanoma cells with high affinity. For this purpose we will use specifically designed retroviral vectors with a SAMEN backbone that are adapted for the transfer of TCR alpha and beta and that in some cases also express the neo(r) gene for G418 selection. In specific aim 2 we will determine the optimal conditions to TCR gene modify human peripheral blood T cells from normal donors and melanoma patients for patient treatment. For this set of experiments, we will use the previously produced A6 and A7 retroviral vectors that carry the low affinity TIL 5 TCR. Using this model, we will optimize methods of transducing and expanding normal PBL. We will evaluate 2 transduction methodologies, spinoculation and retronectin based transduction. We will also optimize methods for in vitro expansion of transduced T-cell populations and study TCR transduction (by RT-PCR) expression (by tetramer staining) as well as functional activity (by Cytokine release and Cr release assay). It is anticipated that methods developed under specific aim 2, will be applicable for cell populations transduced with newer vectors generated under specific aim 1. In specific aim 3 we will determine if TIL 13831 TCR gene modified T cells can be detected and quantified using RTP-CR and competitive RT-PCR. For this purpose specific primere for TIL 13831 will be designed, as well as a clone specific inhibitor. The validity of the test and its accuracy will be extensively evaluated.